CN105586343A

CN105586343A - Torenia-originated promoter capable of acting in petals

Info

Publication number: CN105586343A
Application number: CN201610124827.2A
Authority: CN
Inventors: 田中良和; 胜元幸久
Original assignee: Suntory Holdings Ltd
Current assignee: Suntory Holdings Ltd
Priority date: 2012-02-24
Filing date: 2013-02-19
Publication date: 2016-05-18
Also published as: WO2013125530A1; CA2865206A1; CO7061034A2; RU2014138477A; KR20140125393A; JPWO2013125530A1; EP2818548A4; EP2818548A1; EP3054011A1; CN104254605A; RU2017103447A; US20150020242A1; ECSP14017928A

Abstract

A novel promoter useful for altering the color of flowers of a plant, which is selected from the group consisting of: (1) a nucleic acid which comprises the nucleotide sequence represented by SEQ ID NO: 20, SEQ ID NO: 12 or SEQ ID NO: 21; and (2) a nucleic acid which maintains the same promoter activity as that of the nucleotide sequence represented by SEQ ID NO: 20, SEQ ID NO: 12 or SEQ ID NO: 21, and which comprises a nucleotide sequence that is produced by modifying the original nucleotide sequence by the addition, deletion and/or substitution of one or several nucleotide sequences, or can hybridize with a nucleotide acid comprising a nucleotide sequence complementary to the original nucleotide sequence under highly stringent conditions, or has a 90% or more sequence identity to the original nucleotide sequence.

Description

In petal, bring into play the promoter from blue pig ear of function

The application is that application number is 201380007587.2, and the applying date is on February 19th, 2013, inventionName is called the division of the application for a patent for invention of " promoter from blue pig ear of bringing into play function in petal "Application.

Technical field

The present invention relates to Novel promoter. In more detail, the present invention relates to the flavonoids from blue pig ear3 ', 5 '-hydroxylase (is abbreviated as F3 ' 5 ' H below. ) gene or flavones synzyme (be abbreviated as F belowNS. ) transcriptional control region and the utilization thereof of gene.

Background technology

By utilizing gene recombination technology, useful gene is expressed in object plant, can give it and plantThe proterties that thing is new. So the genetically modified plants of preparation is cultivated by widespread commercial. Because gene expression is adjustedControl is mainly controlled in transcription stage, so transcriptional control is also important on regulate gene expression.In applicable period, applicable tissue, transcribe genes of interest with the intensity being applicable to, to preparation industryUpper useful genetically modified plants is extremely important. Transcribe beginning under many circumstances by being positioned at translational domainThe DNA sequence dna of 5' side control. The initiation site of genetic transcription, its frequency of direct regulation and control will be determinedRegion on DNA is called promoter. Promoter is present in the tens of bp of 5 ' the side place of initiation codon sometimes,Comprise the sequence of TATA box etc. more. It further also has in conjunction with various transcription regulatory factors in 5 ' sideCis-acting elements, the period that the existence control of these cis-acting elements is transcribed, the tissue of transcribing,The intensity of transcribing etc. Transcription regulatory factor is divided into a lot of families according to the difference of its amino acid sequence. ExampleAs, Myb type transcription regulatory factor, bHLH (alkalescence/helix-loop-helix (basichelixloophElix)) type transcription regulatory factor etc. is famous family. In fact, under many circumstances, transcriptional controlRegion and promoter are used on same meaning, there is no strict difference.

Anthocyanin as the main component of pattern is to be generically and collectively referred to as one of secondary metabolite of flavonoids.The color of anthocyanin depends on its structure. The hydroxyl encircling as the chromophoric anthocyanidin B of anthocyaninWhen radix increases, become blue. In representational anthocyanidin, there are delphinidin, anthocyanidin, pelargonidin, the hydroxyl of B ringRadix is respectively 3,2,1. Wherein, delphinidin the most blue (with reference to Fig. 1). In addition, alsoWhile knowing the quantity increase of the aromatic acyl (coumaric acyl, coffee acyl etc.) of modifying anthocyanin, flowerThe color of pigment glycosides becomes blue (being that obtained the maximum absorption moves to long wavelength's direction), and the stability of anthocyanin increasesAdd. Or also known, when the flavonoids of flavones etc. and anthocyanin coexist, color becomes indigo plant and deepens. By this kindEffect is called copigment effect, a kind (referring to non-patent literature 1) that flavones is copigment.The enzyme relevant with the biosynthesis of anthocyanin, the gene of this enzyme of encoding are fully studied (with reference to above-mentionedNon-patent literature 1). For example, catalysis make the enzyme of the reaction that the hydroxyl value of B ring increases be flavonoids 3 '-Hydroxylase (is abbreviated as F3 ' H below. ) and F3 ' 5 ' H. F3 ' H is the synthetic necessary enzyme of anthocyanidin,F3 ' 5 ' H is the synthetic necessary enzyme of delphinidin. Because of the not synthetic delphinidin such as Chinese rose, carnation, chrysanthemum,So, in these plants without blue kind. It is existing by F3 ' 5 ' H gene is expressed in these plants,Thereby make pattern turn blue example (with reference to No. WO1996/036716th, International Publication, following patent documentation4 with No. WO2009/062253). Flavones is synthesized by flavanones by the catalysis of FNS. Existing by makingFlavone synthetase gene in petunia, express and the example that changes pattern (with reference to PlantBiotechnol21377-386(2004))。

And also obtain conclusion for the transcriptional control of the gene of the biosynthetic enzyme of anthocyanin. In placeIn the transcriptional control region of 5 ' side of the initiation codon of these genes, combine Myb type and transcribeThe cis-acting elements sequence of regulatory factor, bHLH type transcription regulatory factor. Known Myb type transcriptional controlSynthetic (the ginseng of the anthocyanin of the factor and bHLH type transcription regulatory factor control petunia, corn, purple perilla etc.According to above-mentioned non-patent literature 1).

The promoter of being responsible for genetic transcription in plant is (below also referred to as transcriptional control region. ) in, haveAll bring into play the so-called constitutive promoter of function in any period of any tissue or stage of development, only spyDetermine the organ-tissue specificity promoter of performance function in organ-tissue and specific in the stage of development onlyThe specificity promoter in period that express period. As for useful gene is expressed at genetically modified plantsPromoter, conventional constitutive promoter. As representational constitutive promoter, there is cauliflower floral leafVirus 35S promoter, on its basis build promoter (referring to non-patent literature 3), Mac1Promoter (referring to non-patent literature 4) etc. But in plant, a lot of genes are organizerOfficial's specificity or period are specific expressed. Its show to make gene carry out tissue and organ specificity or period spyIt is essential to plant that the opposite sex is expressed. Existing utilize this kind of tissue and organ specificity or period specificThe example of the genetic recombination of plant is carried out in transcriptional control region. For example use in addition seed specific transcriptional to adjustThe example that control region accumulates protein in seed.

For bringing into play function at petal internal specific or main promoter and the utilization thereof of bringing into play function in petal,There are reports. For example, existingly use opening from the chalcone synthase gene of toad's-mouth or petuniaMover expresses F3 ' 5 ' H gene and changes the example of petal color in petunia and carnation. AndThe promoter that demonstrates the chalcone synthase gene of Chinese rose has been brought into play function in Chinese rose, chrysanthemum. Also have logicalThe promoter of crossing F3 ' 5 ' the H gene that makes cineraria is expressed and is changed the example of pattern in petunia. ThisAlso promisingly F3 ' 5 ' H gene is expressed in Chrysanthemum Petal and use the flavanones 3-hydroxylation of chrysanthemum outward,The example of enzyme gene promoter.

But this kind of promoter, in object recombinant plant, can be brought into play strong function in which kind of degree,Whether can bring object phenotype, also be difficult to prediction. And, proceed to host plant same or similarWhen base sequence or transgene carry out multicopy or repeat to insert in chromosome, cause sometimes geneSilence (with reference to following non-patent literature 5). Therefore, for multiple exogenous gene expressions are reusedIdentical promoters, causes the silence of gene sometimes, so should avoid. In addition, even if say and flavonoidsSynthetic relevant enzyme gene in petal, bring into play function, the time point of its expression is also different. Known commonFlavones, flavonols, compared with anthocyanin, can express when flower is more early stage. For example,, in order to changeBecome pattern, it is industrially extremely important that acquisition expression time is put different multiple promoters.

Prior art document

Patent documentation

No. WO96/25500th, patent documentation 1 International Publication

No. WO01/72984th, patent documentation 2 International Publications

No. WO94/28140th, patent documentation 3 International Publications

No. WO05/17147th, patent documentation 4 International Publications

Non-patent literature

Non-patent literature 1PlantJ.54,737-749,2008

Non-patent literature 2AgriculturalandBiologicalChemistry, 53,797-800,1989

Non-patent literature 3lantCellPhysiology1996,37,49-59

Non-patent literature 4PlantMolecularBiology1990,15,373-381

Non-patent literature 5AnnalsofBotany1997,79,3-12

Summary of the invention

The problem that the present invention will solve is, is provided for changing the useful Novel promoter of plant pattern.

Present inventor furthers investigate for solving above-mentioned problem, constantly experiment, found that as forChange F3 ' 5 ' H gene and the FNS gene from blue pig ear of the useful Novel promoter of plant patternTranscriptional control region, and confirmed its serviceability, thereby completed the present invention.

Be that the present invention is as follows.

[1] nucleic acid, is characterized in that, is selected from following nucleic acid:

(1) nucleic acid, it is made up of the base sequence shown in sequence number 20,

(2) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And by the base sequence shown in sequence number 20 is carried out one or more base sequences occur additional, disappearance and/ or the modification that replaces after base sequence composition,

(3) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And can be with the nucleic acid being formed by the complementary base sequence of the base sequence shown in sequence number 20 rigorous of heightUnder part, hybridize, and

(4) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And there is at least 90% sequence homogeneity with the base sequence shown in sequence number 20.

[2] expression vector, is characterized in that, comprises the nucleic acid described in described [1].

[3] according to the expression vector described in described [2], it is characterized in that, comprise sequence number 20Shown base sequence.

[4] a non-human host, is characterized in that, by the expression described in described [2] or [3]Carrier transforms.

[5] one Plants or its offspring or their part or tissue, is characterized in that, proceeds to instituteState the nucleic acid described in [1].

[6] according to the plant described in described [5] or its offspring or their part or tissue,It is characterized in that, be cut-flower.

[7] a cut-flower processed goods, is characterized in that, for the cut-flower described in described [6] is used asRaw material and the cut-flower processed goods that obtains.

[8] nucleic acid, is characterized in that, is selected from following nucleic acid:

(1) nucleic acid, it is made up of the base sequence shown in sequence number 12,

(2) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And by the base sequence shown in sequence number 12 is carried out one or more base sequences occur additional, disappearance and/ or the modification that replaces after base sequence composition,

(3) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And can be with the nucleic acid being formed by the complementary base sequence of the base sequence shown in sequence number 12 rigorous of heightUnder part, hybridize, and

(4) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear F3 ' 5 ' H gene,And there is at least 90% sequence homogeneity with the base sequence shown in sequence number 12.

[9] expression vector, is characterized in that, comprises the nucleic acid described in described [8].

[10] according to the expression vector described in described [9], it is characterized in that, comprise sequence number 12Shown base sequence.

[11] a non-human host, is characterized in that, by the table described in described [9] or [10]Reaching carrier transforms.

[12] one Plants or its offspring or their part or tissue, is characterized in that, proceeds toNucleic acid described in described [8].

[13] according to the plant described in described [12] or its offspring or their part or tissue,It is characterized in that, be cut-flower.

[14] a cut-flower processed goods, is characterized in that, for the cut-flower described in described [13] is usedThe cut-flower processed goods of making raw material and obtain.

[15] nucleic acid, is characterized in that, is selected from following nucleic acid:

(1) nucleic acid, it is made up of the base sequence shown in sequence number 21,

(2) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear FNS gene, and by rightBase sequence shown in sequence number 21 carries out one or more base sequences and adds, lacks and/or getBase sequence composition after the modification in generation,

(3) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear FNS gene, and can be withThe nucleic acid being made up of the complementary base sequence of the base sequence shown in sequence number 21 is assorted under the rigorous condition of heightHand over, and

(4) nucleic acid, it can be used as the transcriptional control region performance function of blue pig ear FNS gene, and and orderBase sequence shown in row numbers 21 has at least 90% sequence homogeneity.

[16] expression vector, is characterized in that, comprises the nucleic acid described in described [15].

[17] according to the expression vector described in described [16], it is characterized in that, comprise sequence number 21Shown base sequence.

[18] a non-human host, is characterized in that, by described in described [16] or [17]Expression vector transforms.

[19] one Plants or its offspring or their part or tissue, is characterized in that, proceeds toNucleic acid described in described [15].

[20] according to the plant described in described [19] or its offspring or their part or tissue,It is characterized in that, be cut-flower.

[21] a cut-flower processed goods, is characterized in that, for the cut-flower described in described [20] is usedThe cut-flower processed goods of making raw material and obtain.

[22] expression vector, is characterized in that, comprise the nucleic acid described in described [1] and described in[15] nucleic acid described in.

[23] according to the expression vector described in described [22], it is characterized in that, comprise sequence number 20Base sequence shown in shown base sequence and sequence number 21.

[24] a non-human host, is characterized in that, by described in described [22] or [23]Expression vector transforms.

[25] one Plants or its offspring or their part or tissue, is characterized in that, proceeds toNucleic acid described in nucleic acid described in described [1] and described [15].

[26] according to the plant described in described [25] or its offspring or their part or tissue,It is characterized in that, be cut-flower.

[27] expression vector, is characterized in that, comprise the nucleic acid described in described [8] and described in[15] nucleic acid described in.

[28] according to the expression vector described in described [27], it is characterized in that, comprise sequence number 12Base sequence shown in shown base sequence and sequence number 21.

[29] a non-human host, is characterized in that, by described in described [27] or [28]Expression vector transforms.

[30] one Plants or its offspring or their part or tissue, is characterized in that, proceeds toNucleic acid described in nucleic acid described in described [8] and described [15].

[31] according to the plant described in described [30] or its offspring or their part or tissue,It is characterized in that, be cut-flower.

[32] a cut-flower processed goods, is characterized in that, for the cut-flower described in described [31] is usedThe cut-flower processed goods of making raw material and obtain

Think and in blue pig ear petal, be responsible for the promoter region of enzyme genetic transcription, i.e. control indigo plant by useThe region of pig ear F3 ' 5 ' H gene and FNS genetic transcription, can be long-pending at the flavonoids, anthocyanin of flower etc.In tired tissue, cause specific transcription of foreign genes. As the foreign gene of transcribing, have with pattern,The gene that fragrance is associated, but be not limited to these.

Brief description of the drawings

Fig. 1 is the skeleton diagram of flavonoids biosynthesis pathway.

Fig. 2 is that gene proceeds to the skeleton diagram with binary vector pSPB3797.

Fig. 3 is that gene proceeds to the skeleton diagram with binary vector pSPB3798.

Detailed description of the invention

As transcriptional control of the present invention region, for example, can enumerate by shown in sequence number 20,12 or 21The nucleic acid of base sequence composition. But, also think by passing through the base shown in sequence number 20,12 or 21In the nucleic acid of sequence composition, multiple (1,2,3,4,5,6,7,8,9 or 10) base occurs attachedAdd, lack and/or replace and the promoter of base sequence after modifying composition maintains and original promoterSame activity. Therefore,, as long as bring into play function as transcriptional control region in petal, the present invention also relates toAnd following nucleic acid, it is by the base sequence shown in sequence number 20,12 or 21 is carried out to one or more alkaliBase sequence composition after the modification that basic sequence occurs to add, lack and/or replace.

The present invention also further relates to following nucleic acid, and it can be used as blue pig ear F3 ' 5 ' H gene or FNSThe transcriptional control region performance function of gene, and can with the base sequence shown in sequence number 20,12 or 21Under the rigorous condition of height, hybridize, or can be used as the transcriptional control of blue pig ear F3 ' 5 ' H gene or FNS geneRegion performance function, and have at least 90% sequence with the base sequence shown in sequence number 20,12 or 21Homogeneity.

As these nucleic acid, can enumerate the nucleic acid being formed by following base sequence, can with sequence number 20,12Or the complementary polynucleotide of the base sequence shown in 21 hybridizes under rigorous condition, with sequence number 20,12Or the base sequence shown in 21 is preferably more than approximately 70%, more preferably approximately 80%, 81%, 82%, 83%,84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97%, 98%, most preferably be approximately 99% sequence homogeneity.

At this, rigorous condition refers to easily definite hybridization conditions by those skilled in the art, is generally and complies withRely the empirical condition of probe length, wash temperature and salinity. Conventionally, probe is longer, for being applicable toThe temperature of annealing is higher, and the shorter annealing temperature of probe is lower. Hybridization conventionally depends on complementary strand and is present in and connectsNear but lower than the anneal capability again of time variation DNA in the environment of its fusing point. Particularly, for example, as lowRigorous condition, can be set forth in the washing stage of the rear filter membrane of hybridization, under the temperature conditions of 37 DEG C～42 DEG C,In 5 × SSC, 0.1%SDS solution, wash etc. In addition as the rigorous condition of height, for example, can be set forth in,Washing stage, in 65 DEG C, 0.1 × SSC and 0.1%SDS solution, wash etc. By rigorous condition is carriedHigh to higher, can obtain the much higher nucleotides of sequence homology or homogeneity.

The present invention also relates to the transcriptional control region that comprises blue pig ear F3 ' 5 ' H gene and/or FNS geneCarrier, and the non-human host who is transformed by this carrier.

And, the present invention relates to the transcription regulatory region of blue pig ear F3 ' 5 ' H gene and/or FNS geneTerritory is connected with useful foreign gene, proceed to this foreign gene and obtain there is the color etc. of change serviceabilityThe plant of shape or its offspring or their part or tissue, this part also can be cut-flower. As turningThe example of the plant changing, can enumerate Chinese rose, chrysanthemum, carnation, toad's-mouth, cyclamen, orchid, largeBeautiful flower, Lisianthus, freesia, African Chrysanthemum, gladiolus, rosy clouds grass, jonquil, lily, fish pelargonium,Geranium wilfordii, petunia, blue pig ear, tulip, rice, morning glory, barley, wheat, vegetable seed, horseBell potato, tomato, white poplar, banana, blue gum, sweet potato, soya bean, clover, lupin, corn etc.,But be not limited to these.

The present invention relates to use the processed goods (cut-flower processed goods) of above-mentioned cut-flower. At this, process as cut-flowerProduct, comprise dry petal, preserved flower, dried flower, resin enclosed article of using this cut-flower etc., but are not limited to thisA bit.

Embodiment

Below, by embodiment, illustrate the present invention.

Without particular/special requirement in the situation that, molecular biological method is according to MolecularCloning (SAmbrookandRussell, 2001) carry out. The conversion of Agrobacterium, petunia, Chinese rose is as international publicOpen No. WO2004/020637 or patent documentation 4 in record, but be not limited to these methods.

[embodiment 1: the clone in the transcriptional control region of blue pig ear F3 ' 5 ' H gene TBG10]

The base sequence of blue pig ear F3 ' 5 ' HcDNA be known (MolecularBreeding6,239-246 (2000), GeneBankDNA preserving number AB012925). By λ DASHII (AgilentScience and technology (AgilentTechnologies) Co., Ltd.) as carrier, the side of recommending by producerMethod builds the chromosomal dna library of blue pig ear. Blue pig ear chromosomal DNA is from blue pig ear kind SummerWIn the leaf of aveBlue (Suntory Flowers Ltd.), prepare. By obtained blue pig ear chromosome DNA library usage flag blue pig ear F3 ' 5 ' HcDNA screening, reclaim and blue pig ear F3 ' 5 ' HcThe plaque of the bacteriophage of DNA hybridization. Taking this bacteriophage as template, with 2 kinds of oligonucleotides (T3pro:5 '-AATTAACCCTCACTAAAGGG-3 ' (sequence number 1), T7pro:5 '-TAATACGACTCACTATAGGG-3 ' (sequence number 2)) for primer carries out PCR, by the order comprising from the chromosomal DNA of blue pig earThe DNA fragmentation of row increases. Taking this DNA as template, with one group of oligonucleotides (T3pro, THF2RV:5 '-CTATGGAAGATAACAATG-3 ' (sequence number 3)) or one group of oligonucleotides (T7pro, THF2RV) for primer carries out PCR. By increase with the PCR that can use T3pro and THF2RV approximately 5.6The DNA fragmentation (sequence number 4) of kb is cloned on pCR2.1TOPO (invitrogen company). InstituteThe plasmid obtaining is as pSPB3745.

Taking above-mentioned bacteriophage as template, with one group of oligonucleotides (TBG10proFW:5 '-TGAAATATAAATATGAATGGG-3 ' (sequence number 5), TBG10proRV:5 '-ACTGAATGGTGACTAGCTGC-3 ' (sequence number 6)) carry out PCR for primer. Obtained DNA fragmentation is cloned into BluntII-TOPO (invitrogen company) is upper, as plasmid pSPB3764 (comprising sequence number 7). This DNA orderRow are as shown in sequence number 7. And then taking pSPB3764 as template, with one group of oligonucleotides (TBG10proFW, TBG10proXbaIRV:5 '-TCTAGACTGAATGGTGACTAGC-3 ' (sequence number 8)) be primerCarry out PCR. Upper by this DNA fragmentation being cloned into BluntII-TOPO, obtain pSPB3770. At thisOn plasmid, comprise the 5 ' non-translated sequence of the blue pig ear F3 ' 5 ' H of about 4kb, hold restricted property 3 'The recognition sequence of restriction endonuclease XbaI.

[embodiment 2: the clone in the transcriptional control region of blue pig ear F3 ' 5 ' H gene TBG16]

Plaque that obtain in propagation embodiment 1 and bacteriophage blue pig ear F3 ' 5 ' HcDNA hybridization,Prepare phage DNA. As template, with one group of oligonucleotides (T3pro, THF2RV) or one group of widowNucleotides (T7pro, THF2RV) carries out PCR for primer. Will be from the P from (T3pro, THF2RV)It is upper that the DNA fragmentation of the approximately 2.3kb obtaining in CR is cloned into pCR2.1TOPO, as pSPB3746. ShouldSequence is as shown in sequence number 9. Taking this plasmid as template, with one group of oligonucleotides (TBG16proFW:5 '-TCCTATTGCACTCGTTTTTTC-3 ' (sequence number 10), TBG16proRV:5 '-ACTGAATGGTGACTAGCCGC-3 ' (sequence number 11)) carry out PCR for primer. Obtained DNA fragmentation is cloned into BlUntII-TOPO (invitrogen company) is upper, as plasmid pSPB3758. In this plasmid, compriseDNA sequence dna is as shown in sequence number 12. And then taking pSPB3758 as template, with one group of oligonucleotides (TBG16proFW, TBG16proBamHI:5 '-GGATCCACTGAATGGTGACTAGCC-3 ' (sequence number 13))For primer carries out PCR. Upper by this DNA fragmentation being cloned into BluntII-TOPO, obtain pSPB3768. In this plasmid, the 5 ' non-translated sequence of the blue pig ear F3 ' 5 ' H that comprises about 0.7kb, 3 'End has the recognition sequence of restriction enzyme BamHI.

[embodiment 3: the clone in the transcriptional control region of blue pig ear FNS gene]

By the blue pig ear chromosomal dna library usage flag obtaining in embodiment 1 blue pig ear FNScDNA screening, reclaims the plaque of the bacteriophage hybridizing with blue pig ear FNScDNA. Biting of propagation bacteriophageBacterial plaque, prepares phage DNA. Taking this DNA as template, with one group of oligonucleotides (T3pro, TFNSR3:5 '-ATTCCTAATGGGCTGAAAGTG-3 ' (sequence number 14)) or one group of oligonucleotides (T7pro, TFNSR3) be primer, carry out PCR. By approximately 4.2 of the pcr amplification of available use T7pro and TFNSR3The DNA fragmentation (sequence number 15) of kb is cloned on pCR2.1TOPO. The plasmid obtaining is as pSP B3747。

Taking above-mentioned phage DNA as template, with one group of oligonucleotides (TFNS1proFW:5 '-CAAATGAAACCCCATCAGTGTC-3 ' (sequence number 16), TFNS1proRV:5 '-GCTTTATATATATTTTTTTAGCGC-3 ' (sequence number 17)) be primer, carry out PCR. Increased DNA fragmentation is cloned into BlunTII-TOPO is upper, as plasmid pSPB3759. Insert sequence in this plasmid as shown in sequence number 18.Taking pSPB3759 as template, with one group of oligonucleotides (TFNS1proFW, TFNS1pBamHIRV:5 '-GGATCCGCTTTATATATATTTTTTTAGC-3 ' (sequence number 19)) be primer, carry out PCR. Pass throughThis DNA fragmentation is cloned into BluntTOPO above, obtains pSPB3769. In this plasmid, comprise approximately 3.65 ' the non-translated sequence of the blue pig ear FNS of kb, has the knowledge of restriction enzyme BamHI at 3 ' endOther sequence.

[embodiment 4: the binary vector in the transcriptional control region that comprises blue pig ear F3 ' 5 ' H and FNSPreparation]

Cut the plasmid pSPB3770 obtaining in embodiment 1 and carry out end smoothing with SspI enzyme, entering oneStep XbaI enzyme cutting. By the DNA fragmentation of obtained 1.6kb (sequence number 20) with use HindIIIEnzyme is cut pBinPLUS and is carried out end smoothing, and further connects with the DNA fragmentation of XbaI enzyme cutting,As plasmid pSPB3791.

Will be with BamHI and PacI digested plasmid pSPB580 (as patent documentation 4 is recorded) and the D obtainingNA fragment (comprising F3 ' 5 ' HBP40cDNA and petunia D8 terminator sequence from heartsease)The pBinPLUS cutting with BamHI and PacI enzyme and obtain is connected, and obtains plasmid pSPB3795. Pass throughBy the DNA fragmentation obtaining with XbaI and PacI digested plasmid pSPB3791 with XbaI and PacIEnzyme is cut pSPB3795 and the DNA fragmentation that obtains connects, and obtains binary vector pSPB3793. At this double baseOn carrier, from the transcriptional control region of TBG10, from the F3 ' 5 ' HcDNA of heartsease, short leadingOx D8 terminator is linked in sequence.

Cut the plasmid pSPB3768 obtaining in embodiment 2 and carry out end smoothing with EcoRI enzyme, entering oneStep is cut with BamHI enzyme. By the DNA fragmentation of obtained 0.7kb with cut pBinPLUS with HindIII enzymeAnd carrying out end smoothing, the DNA fragmentation of further cutting with BamHI enzyme connects, as plasmid pSPB3790. By the DNA fragmentation of cutting above-mentioned plasmid with BamHI and PacI enzyme and obtain and use BamHI and PacIEnzyme is cut pSPB580 and the DNA fragmentation that obtains connects, and obtains binary vector pSPB3792. Carry at this double baseOn body, from the transcriptional control region of TBG16, from F3 ' 5 ' HcDNA, the petunia D of heartsease8 terminators are linked in sequence.

Cut the plasmid pSPB3769 obtaining in embodiment 3 and carry out end smoothing with SpeI enzyme, entering oneStep is cut with BamHI enzyme. By the DNA fragmentation of obtained 1.4kb (sequence number 21) with use HindIII enzymeCut pSPB3383 and carry out end smoothing, the DNA fragmentation of further cutting with BamHI enzyme connects, and obtainsPlasmid pSPB3789. On this plasmid, blue pig ear FNS transcriptional control region, blue pig ear FNScDNA,Petunia D8 terminator is linked in sequence, can be by the DNA fragmentation that comprises these is come with AscI cuttingReclaim. The binary vector of AscI that this DNA fragmentation is proceeded to pSPB3793 is as pSPB3798 (referenceFig. 3), and the binary vector of AscI that proceeds to pSPB3792 as pSPB3797 (with reference to Fig. 2).

[embodiment 5: the expression (result of PT315) in petunia]

The double base plasmid pSPB3797 or the pSPB3798 that in embodiment 4, record are proceeded to Agrobacterium tumefaciems ((AGrobacteriumtumefaciens) in system Ag10. Use this conversion Agrobacterium, conversion is opened light pinkThe petunia system Skr4xSw63 (as International Publication is recorded for No. WO93/01290) of look flower. Will be from pThe test block of SPB3797 is as PT314, using the test block from pSPB3798 as PT315. ObtainThe transformant of 2 systems independently in PT314. The pattern of two systems becomes aubergine. Following table 1 and table 2Middle expression respectively analyzed the anthocyanidin of the petal of PT314 and PT315 and Skr4xSw63 known methodResult.

Table 1

Sample name	Delphinidin	Petunidin	Pelargonidin	Peonidin	Malvidin
						PT314-1	5.4	98.6	3.8	20.6	1950
PT314-2	4.2	76.6	3.3	17.6	1150
						Skr4xSw63	0	2.9	22.5	15.8	60.3

The μ g/g of unit petal fresh weight

Obtain in PT315 the independently transformant of 26 systems. Wherein, the pattern of 15 systems becomes purplish redLook. The anthocyanidin of the petal of these a part of system and Skr4xSw63 and flavones are divided with known methodAnalyse.

Table 2

Sample name	Delphinidin	Petunidin	Pelargonidin	Peonidin	Malvidin	Apiolin	Quercetin
								PT315-1	3.0	72	0	2.7	1330	2.8	11
PT315-9	6.8	126	0	0.6	1580	1.9	31
								PT315-12	2.0	33	1.0	7.0	393	44	9.4
PT315-13	3.0	72	0	2.8	1550	4.9	17
								PT315-14	4.0	72	0	5.3	1290	5.6	11
PT315-15	5.0	109	0	1.0	1630	4.6	30
								PT315-17	5.6	116	0	8.2	1690	18	27
PT315-19	1.9	39	1.5	11	756	3.0	7.0
								PT315-20	3.6	93	0	4.8	1190	3.3	17
PT315-21	10	185	0	14	1130	1.4	8.1
								PT315-25	5.8	101	0	0.6	1540	2.4	11
PT315-26	3.3	71	0	1	862	3.5	25
								Skr4xSw63	0	3.2	5.6	11	74	0	0

Unit: μ g/g petal fresh weight

Above result shows, the transcriptional control region from TBG10 that comprises pSPB3770, comprises pThe transcriptional control region from TBG16 of SPB3768, the tune of transcribing from FNS that comprises pSPB3759Function is brought into play in control region in petunia not of the same race.

[embodiment 6: to turning of the pSPB3798 in modern rose cultivars " song (OceanSong) of ocean "Enter]

The double base plasmid pSPB3798 recording in embodiment 4 is proceeded to Agrobacterium tumefaciems (AgrobacteriUmtumefaciens) in system Ag10. Use this conversion Agrobacterium, transform modern rose cultivars " ocean itSong (OceanSong) ", obtain the transformant of 15 individualities. Carry out in 15 individualities of anthocyanidin analysis,The delphinidin of can having confirmed 5 individuals accumulated. Delphinidin containing ratio is up to 7.3% (average 5.0%).In addition, the result that flavonols flavones is analyzed, detects myricetin, 7 in 8 individualities in 9 individualitiesIn individuality, detect quercetin. Delphinidin, myricetin, quercetin all can be by from heartseasesThe effect of F3 ' 5 ' H generates, and shows the transcriptional control region from blue pig ear TBG10 gene in Chinese rosePerformance function.

On the other hand, because all can newly confirming there is flavones in whole individualities of analyzing (quercetin,Cyanidenon, apiolin) accumulation, so, show the transcriptional control from the FNS gene of blue pig earFunction has been brought into play in region in Chinese rose. In addition, flavones total amount is up to 0.658 in every 1g petal fresh weight8mg。

The assay value of representational transformant as shown in the following Table 3.

Table 3

Del: delphinidin, Cya: anthocyanidin, Pel: pelargonidin, M: myricetin, Q: Quercetin,

K: Kaempferol, Tri: quercetin, Lut: cyanidenon, Api: apiolin,

Del (%): the ratio of delphinidin in total anthocyanidin

[embodiment 7: to turning of the pSPB3797 in modern rose cultivars " song (OceanSong) of ocean "Enter]

Proceed to embodiment 4 in the modern rose cultivars " song (OceanSong) of ocean " of rattan colour system in, recordPSPB3797, obtain the transformant of 7 individualities. In whole 7 individualities that carry out anthocyanidin analysis, notConfirm the accumulation of delphinidin. In addition, the result that flavonols flavones is analyzed, does not exist and can detect poplarThe individuality of syphilis, quercetin. The above results shows, from the TBG16 gene of blue pig ear in Chinese rosePromoter is not brought into play function.

On the other hand, by the FNS effect from blue pig ear, all can newly confirm to carry out the institute analyzingThere is the accumulation that has flavones (cyanidenon, apiolin) in individuality, so, the FN from blue pig ear shownThe promoter of S gene has been brought into play function in Chinese rose. In addition, flavones total amount in every 1g petal fresh weightHeight is 0.1704mg.

The assay value of representational transformant as shown in the following Table 4.

Table 4

K: Kaempferol, Tri: quercetin, Lut: cyanidenon, Api: apiolin,

Del (%): the ratio of delphinidin in total anthocyanidin

Known think in the spending of blue pig ear, be responsible for the promoter region of enzyme genetic transcription, the i.e. class of blue pig earThe transcriptional control region of flavones 3 ', 5 '-'-hydroxylase gene and flavone synthetase gene is short leading not of the same raceIn ox and Chinese rose petal, bring into play function as transcriptional control region. Therefore, utilize these transcription regulatory regionsTerritory, can be in the accumulation of flower etc. in the tissue of anthocyanin, cause the specific transcriptional of foreign gene. AsThe foreign gene of transcribing, has the gene being associated with pattern, fragrance, but is not limited to these.

Claims

1. a nucleic acid, is characterized in that, is selected from following nucleic acid:

2. an expression vector, is characterized in that, comprises the nucleic acid described in claim 1.

3. according to the expression vector described in claim 2, it is characterized in that, comprise 21 of sequence numbersThe base sequence showing.

4. the non-human host's of genetic recombination a preparation method, is characterized in that, comprises and passes through claimExpression vector described in 2 or 3 and the step that transforms.

5. a preparation method for genetically modified plants or its offspring or their part, is characterized in that,Comprise the step that proceeds to the nucleic acid described in claim 1.

6. according to the system of the genetically modified plants described in claim 5 or its offspring or their partPreparation Method, is characterized in that, the part of described genetically modified plants is tissue.

7. according to the system of the genetically modified plants described in claim 5 or its offspring or their partPreparation Method, is characterized in that, it is for making cut-flower.

8. a cut-flower processed goods, is characterized in that, it is to be selected to pass through described in claim 5The preparation method of genetically modified plants or its offspring or their part and the cut-flower made as raw material andThe dry petal, preserved flower, dried flower or the resin enclosed article that obtain.